HIV viral markers (e.g., p24 antigen, reverse transcriptase(rt) activity) currently serve as surrogates for estimating viral load and clinical prognosis. Sensitivity of methods for isolating and recovering infectious virus is limited and does not always correlate with viral markers. This project is aimed at developing extremely sensitive assays for detection, isolation, and quantitation of infectious HIV and rare infected cells from a predominantly uninfected cell background (i.e., blood). Our initial goal is to develop and qualify these ultra sensitive techniques and then use them for 1) evaluating the number of infected cells and the infectious virus load within specific blood cell subsets, 2) determining the infectivity of different patient isolates, and 3) defining benchmarks for evaluation of viral marker assays. This work will serve as the foundation for studies planned to evaluate viral load and its impact on prognosis, infectivity of human samples, and efficacy of antiviral therapy.